Most of the therapeutic applications of pharmaceuticals benefit from maintaining a therapeutic effective concentration over a prolonged period of time, often requiring a frequent administration or infusions, or a loco-regional application or subcutaneously of the drug utilizing a slow adsorption into the blood stream in order to maintain an effective concentration over a prolonged period of time. When a drug is administered by rapid intravenous injection into the vascular system, its removal from the blood almost always occurs in a biphasic fashion (see Greenblatt (1985) Ann. Rev. Med. 36:421-427). This can be mathematically described by a two-compartment model, which resolves the body into a central compartment and a peripheral compartment (see Dhillon and Gill: Basic Pharmacokinetics). These compartments have no distinct physiological or anatomical delimitation, however, the central compartment is considered to comprise tissues that are highly perfused (e.g. heart, lungs, kidneys, liver and brain) whilst the peripheral compartment comprises less well-perfused tissues (e.g. muscle, fat and skin). A two-compartment model assumes that upon drug administration into the central compartment, e.g. into the blood stream, the drug distributes between the central compartment and the peripheral compartment. However, the drug does not achieve instantaneous distribution, i.e. equilibration, between the two compartments. The drug concentration-time profile shows a curve, with the log drug concentration-time plot showing a biphasic response which can be used to distinguish whether a drug shows a one- or two-compartment model (see Dhillon and Gill: Basic Pharmacokinetics). Immediately after the dose is given, there is a phase of rapid drug disappearance from the blood, usually lasting from a few minutes to an hour or two, which may lead to a very substantial decrement in drug concentrations in blood. This initial phase (described by the initial or distribution plasma half-life; t1/2α) of rapid drug disappearance is determined mainly by reversible distribution of drug out of the “central” compartment, of which the vascular system is a component, into storage sites in peripheral tissues; very little of this initial rapid decline is determined by elimination or clearance. After distribution is complete, the blood concentration curve enters a less rapid phase of drug disappearance, termed the elimination phase (described by the terminal or elimination plasma half-life; t1/2β), during which drug disappearance is determined mainly by irreversible clearance. The pattern of drug decline during this elimination phase is used to calculate the elimination plasma half-life which is generally determined only after drug distribution equilibrium has been attained (see Greenblatt (1985) Ann. Rev. Med. 36:421-427). Both, the initial plasma half-life and the terminal plasma half-life of a substance, e.g. a pharmaceutical, can be influenced in order to extend the bioavailability of such substance in the body by preventing its rapid clearance from the blood.
Small molecule pharmaceuticals, in particular most small protein therapeutics, including many of the alternative recombinant antibody formats (Kontermann (2010) Curr. Opin. Mol. ther. 12:176-183) but also the emerging class of alternative scaffold proteins (Nuttall & Walsh (2008) Curr. Opin. Pharmacol. 8:609-615; Gebauer & Skerra (2009) Curr. Opin. Chem. Biol. 13:245-255), suffer from a short serum half-life mainly due to their rapid clearance from circulation (Batra et al. (2002) Curr. Opin. Biotechnol. 13:603-608). These limitations of small size drugs has led to the development and implementation of half-life extension strategies to prolong circulation of these recombinant antibodies in the blood and thus to improve administration and pharmacokinetic as well as pharmacodynamic properties.
Extension of the half-life can help to reduce the number of applications and to lower doses, thus are beneficial for therapeutic but also economic reasons. Strategies to extend the plasma half-life of pharmaceuticals and therapeutic proteins have, therefore, attracted increasing interest (Pisal et al., (2010) J. Pharmaceut. Sci. 99:2557-2575; Kontermann (2009) BioDrugs 23:93-109; Kontermann (2011) Curr. Opin. Biotechnol. in press).
Several mechanisms are involved in clearance of drugs from circulation including peripheral blood-mediated elimination by proteolysis, renal and hepatic elimination, and elimination by receptor-mediated endocytosis (Tang et al. (2004) J. Pharmaceut. Sci. 93:2184-2204). Molecules possessing a small size, i.e. a low molecular mass with a threshold in the range of 40-50 kDa, are rapidly cleared by renal filtration and degradation. Responsible for renal clearance is the glomerular filtration barrier (GBM) formed by the fenestrated endothelium, the glomerular basement membrane and the slit diaphragm located between the podocyte foot processes (Tryggvason & Wartiovaara (2005) Physiology 20:96-101). While the fenestrae between the glomerular endothelial cells are rather large (50-100 nm) allowing free diffusion of molecules, the slit diaphragm represents the ultimate macromolecular barrier, forming an isoporous, zipper-like filter structure with numerous small, 4-5 nm diameter pores and a lower number of 8-10 nm diameter pores (Haraldsson & Sirensson (2004) New Physiol. Sci. 19:7-10; Wartiovaara et al. (2004) J. Clin. Invest. 114:1475-1483). Molecules with a hydrodynamic radius smaller than approximately 4-5 nm are therefore rapidly cleared from the blood. In addition, the charge of a protein contributes to renal filtration. Proteoglycans of the endothelial cells and the GBM form an anionic barrier, which partially prevents the traversal of negatively charged plasma macromolecules (Tryggvason & Wartiovaara (2005) Physiology 20:96-101). Consequently, the size of a protein therapeutic, i.e. its hydrodynamic radius, but also its physicochemical properties represent starting points in order to improve half-life. Furthermore, some plasma proteins such as serum albumin and IgG molecules possess an extraordinary long half-life in the range of 2-4 weeks in humans, which clearly discriminates these molecules from all the other plasma proteins (Kontermann (2009) BioDrugs 23:93-109). Responsible is a recycling through the neonatal Fc receptor (FcRn, Brambell receptor) (Roopenian & Akilesh (2007) Nat. Rev. Immunol. 7:715-725). Albumin and IgGs taken up by cells, e.g. endothelial cells, through macropinocytosis bind to the FcRn in a pH-dependent manner in the acidic environment of the early endosome. This binding diverges albumin and IgG from degradation in the lysosomal compartment and redirects them to the plasma membrane, where they are released back into the blood plasma due to the neutral pH. This offers additional opportunities to extend or modulate the half-life of proteins, e.g. through fusion to albumin or the Fc-region of IgG (Kontermann (2009) BioDrugs 23, 93-109). Finally, protein drugs that bind to a cellular surface receptor will be internalized by receptor-mediated endocytosis and subjected to lysosomal degradation if the protein drug stays bound to the receptor (Tang et al. (2004) J. Pharmaceut. Sci. 93:2184-2204; Lao & Kamei (2008) Biotechnol. Prog. 24:2-7). Hence, engineering of the interaction of the therapeutic protein with its receptor(s) at acidic pH can therefore also prolong half-life of the protein by allowing recycling of the unbound molecules into the blood stream as shown for engineered G-CSF and an anti-IL6 receptor antibody (Sarkar et al. (2002) Nat. Biotechnol. 20:908-913; Igawa et al. (2010) Nat. Biotechnol. 28:1203-1208).
Several half-life extension strategies have been developed in recent years (Kontermann (2009) BioDrugs 23:93-109; Kontermann (2011) Curr. Opin. Biotechnol. in press), including strategies such as PEGylation and hyperglycosylation with the aim to increase the hydrodynamic volume of the protein to reduce renal clearance, as well as strategies utilizing recycling processes executed by the neonatal Fc receptor (FcRn), which is responsible for the extraordinary long half-lives of serum IgGs and of serum albumin (Kim et al. (2006) Clin. Immunol. 122:146-155). For example, albumin has been employed for half-life extension through the generation of albumin fusion proteins. Several albumin fusion proteins, e.g. albinterferon alfa-2b and a coagulation factor IX-HSA fusion protein, have already entered clinical trials (Nelson et al. (2010) Gastroenterology 139:1267-1276; Metzner et al. (2009) Thromb. Haemost. 102:634-644). In addition, various molecules exhibiting albumin-binding activity have been used for half-life extension. For this approach, the albumin-binding moiety is coupled or fused to the therapeutic protein leading to reversible binding to serum albumin after administration. Such albumin-binding molecules include fatty acids, organic molecules, peptides, single-chain Fv, domain antibodies, nanobodies but also domains from naturally occurring proteins capable of binding albumin (for review see: Kontermann (2009) BioDrugs 23:93-109). For example, an albumin-binding domain (ABD) from streptococcal protein G was used to prolong the plasma half-life of recombinant antibodies and Affibody molecules (Stork et al. (2007) Protein Eng. Des. Sel. 20:569-576; Andersen et al. (2010) J. Biol. Chem. 286:5234-5241). Fusion of the ABD resulted in similar half-lives as seen for an albumin fusion protein and an improved tumor accumulation as shown for a bispecific single-chain diabody (Stork et al. (2007) Protein Eng. Des. Sel. 20:569-576; Stork et al. (2009) J. Biol. Chem. 284:25612-25619). These studies, however, also revealed that albumin and ABD fusion proteins do not reach the long half-life of IgG molecules. Attempts to further prolong half-life by applying an ABD with increased affinity for albumin resulted only in a marginal improvement (Hopp et al., 2010, Protein Eng. Des. Sel. 23:827-834). Non-covalent interaction with serum IgG also represents a feasible alternative to binding to serum albumin. This approach was already tested with a bispecific diabody with affinity for mouse Fcγ1, which prolonged the terminal plasma half-life of the diabody from 1.7 h to 10 h in mice (Holliger et al. (1997) Nat. Biotechnol. 15:632-636).
However, many disadvantages are associated with above strategies of extending the plasma half-life of pharmaceuticals. The usage of PEG, polysialic acid and HES requires their chemical conjugation to the pharmaceutical, which consequently complicates the production and analysis of the final product. PEG is not biologically degradable and may accumulate in the body of a patient which may lead to further complications. Moreover, it has been shown that these modification were only able to prolong the serum half-life of pharmaceuticals to a limited extend. Similarly, also the serum half-life extension via the conjugation, fusion or binding of the pharmaceutical to serum albumin or via Fc-fusion proteins remains significantly below the serum half-life of IgG. There is thus, a clear need for the development of new strategies allowing for the extension of the serum half-life of pharmaceuticals, especially of therapeutic proteins, which overcome these disadvantages.
Present inventors surprisingly found that the fusion of pharmaceuticals to an immunoglobulin-binding domain (IgBD) solves this problem. IgBDs are known from various bacterial proteins, e.g. staphylococcal protein A (SpA), streptococcal protein G (SpG) and protein L of Peptostreptococcus (PpL) (Tashiro & Montelione (1995) Curr. Biol. 5:471-481; Sidorin & Solov'eva (2011) Biochemistry (Mosc.) 76:363-378). These IgBDs have a length of 50 to 60 amino acid residues and form either a 3-α-helix bundle or a compact structure composed of a 4-stranded β-sheet and one α-helix (Tashiro & Montelione (1995) Curr. Biol. 5:471-481). IgBDs are, thus, particularly stable which benefits the production and storage properties of fusion proteins comprising them.
IgBDs show a high affinity to serum immunoglobulins, with most of them binding to the same location on the Fc domain of an immunoglobulin as the neonatal Fc receptor, e.g. in IgG the primary binding site is located at the CH2-CH3 interface of one heavy chain (Deisenhofer (1981) Biochemistry 20:2361-2370). They are thus, competing with the FcRn binding and may negatively influence the recycling of the immunoglobulin molecule via the FcRn. For these reasons, IgBDs have so far not been considered for the extension of the serum half-life of pharmaceuticals. However, some bacterial IgBDs are also capable of binding to different regions of the Fab fragment (Tashiro & Montelione (1995) Curr. Biol. 5:471-481). Present inventors were able to show that the fusion of pharmaceuticals to an IgBD significantly prolongs the serum half-life of such pharmaceutical, probably due to the fact that these IgBDs do not compete with the Fc receptor binding.
The fusion or conjugation of a pharmaceutical to such IgBD thus, represents an advantageous possibility of extending their serum half-life as the binding of such fusion protein to an immunoglobulin molecule has a twofold effect; firstly, the clearance by renal filtration and degradation is limited or prevented, and secondly, the recycling of the fusion protein via the FcRn is allowed for.
The complexes of the present invention provide inter alia the following advantageous properties increase of the solubility of the respective pharmaceutically active moiety in vivo, increase of the in vitro stability of the respective pharmaceutically active moiety, which results in an extended shelf-life of such fusion protein. In cases wherein the pharmaceutical active moiety is a protein or peptide, a further advantage of fusing such moiety to an IgBD of the present invention is the increased expression of such fusion proteins, e.g. in mammalian expression systems. In addition, the complexing of the pharmaceutical moiety to an immunoglobulin binding moiety, in particular if the pharmaceutical moiety is a protein or peptide allows an easier and/or faster purification of such pharmaceutical.